Online Issues

Journal Vol 47 (4)

<< All Back-issues

<< This Issue's Table of Contents

Selection, Acclimation, Training, and Preparation of Dogs for the Research Setting

LaVonne D. Meunier

LaVonne D. Meunier, D.V.M., DACLAM, is Director, Veterinary Medicine, Department of Laboratory Animal Science, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA.

Address correspondence and reprint requests to Dr. Meunier, GlaxoSmithKline Pharmaceuticals, Mail Stop UW2630, P.O. Box 1539, King of Prussia, PA 19406-0939, or email LaVonne.D.Meunier@gsk.com.

Abstract

Dogs have made and will continue to make valuable contributions as animal models in biomedical research. A comprehensive approach from time of breeding through completion of in-life usage is necessary to ensure that high-quality dog models are used in studies. This approach ensures good care and minimizes the impact of interanimal variability on experimental results. Guidance related to choosing and developing high-quality laboratory dogs and managing canine research colonies is provided in this article. Ensuring that dogs are healthy, well adapted, and cooperative involves good communication between vendors, veterinarians, care staff, and researchers to develop appropriate dog husbandry programs. These programs are designed to minimize animal stress and distress from the postweaning period through the transfer and acclimation period within the research facility. Canine socialization and training programs provided by skilled personnel, together with comprehensive veterinary health programs, can further enhance animal welfare and minimize interanimal and group variability in studies.

Key Words: acclimation; colony management; dog; research; restraint; socialization; stress; training

Introduction

In biomedical research it is well recognized that individual animal variability can affect study results. To minimize animal variability, researchers should be provided with normal, healthy, well-adapted animals that represent a physiological model characterized by a narrow range of physical, behavioral, and clinical parameters. Accomplishing this goal can be challenging, even when using the dog, a species that has been well domesticated and generally well adapted to research environments. Maximizing successful experimental outcomes in studies with dogs involves ensuring that the animals are healthy, providing a suitable environment, minimizing stress, and preventing or alleviating distress. This comprehensive approach involves well-planned and well-managed colony health and care programs that include ongoing evaluation of animals before, during, and after experimental study.

Many husbandry, environmental, and experimental factors can trigger stress responses in research dogs that may disrupt their homeostasis. The degree of physiological disruption, and ultimately the impact on the animal and the study, depends on a variety of factors such as intensity, duration, and novelty of stimulus. Because stress inducers can significantly affect a dog's well-being and experimental outcomes due to neuroendocrine effects (Clark et al. 1997a), it is important to anticipate the stressful experiences to which the dogs may be exposed and the procedures they will undergo at both the vendor and the research facility. The goal of a comprehensive dog care program is to develop appropriate socialization, acclimation, and training regimens that minimize stress and distress, , thereby leading to improvements in the quality of scientific data (Boxall et al. 2004).

Acquisition and Source Selection

Dogs are good animal models for many studies based on their size, biological features, and acceptable behavioral characteristics (e.g., cooperative, docile nature) (Dysko et al. 2002). However, investigators must be certain that the justification of dogs as the species of choice is appropriate. The choice of dog used in research and testing should be based on considerations of characteristics that produce the best science when combined with optimal animal welfare (BVAAWF/FRAME/RSPCA/UFAW 2004b).

In the United States, facilities can purchase dogs through Class A or B dealers. Class A dealers breed and raise dogs on their own premises in closed or stable colonies. Class B dealers are involved in the purchase and/or resale of animals (CFR 1985). Beagles are the most commonly bred dogs for biomedical research, although other breeds (e.g., foxhounds) are also commercially bred for use in research (Dysko et al. 2002). Dogs from Class A dealers have known genetic backgrounds and usually have had limited exposure to infectious diseases; they are referred to as purpose-bred when they have been bred specifically for research. Random-source dogs are obtained from any source that did not breed and raise the animals on their premises. These dogs can be obtained from pounds or shelters and have unknown or incomplete medical histories.

Purchasing purpose-bred dogs affords the buyer opportunities to perform site visits to the breeding facility to evaluate the animals (e.g., general health, genetics-based concerns, behavioral characteristics) and the facilities; to review relevant documentation (e.g., standard operating procedures); and to obtain other salient information. It is important to scrutinize vendors who sell dogs for use in studies conducted under the Good Laboratory Practice Act (GLPs¹) to ensure that they follow appropriate procedures and fulfill regulatory requirements. Site visits aid in the determination of the breeder's commitment to appropriate animal husbandry programs, to the animals' adequate preparation for future experimentation, and to any additional screening, behavioral management, and diagnostic evaluations that should occur before shipment or at the end-user's facility.

Vendor Health and Genetic Considerations

Researchers and veterinary staff should clearly communicate their expectations to the vendor regarding the type and health status of the dog needed. Animal criteria or specifications include sex, age, body weight range, and size as well as the identification of potentially unacceptable physical or behavioral characteristics. One approach is for breeding facility and veterinary staff to partner in establishing guidelines on colony management and screening procedures at the vendor site (e.g., vaccination requirements, preshipment clinical pathology and electrocardiograms (ECGs¹), acclimation to slings), which help fulfill the needs and regulatory requirements at the institution. Some vendors offer animals considered to be “seconds” for sale that may not meet criteria for some institutions but will be acceptable by others. If it is desirable to have specific pathogen-free (SPF¹) animals, then the veterinary staff should determine which pathogens or opportunists are unacceptable, and should ensure that the breeding facility can meet the SPF criteria.

Breeder source and parentage are a very important part of determining animal qualities. Breeders should identify and document dogs in the colony that are considered prime candidates for breeding stock and exclude those with health and behavioral problems, especially if there is a genetic basis in such cases. Beagles are commonly bred for research purposes because of their good behavioral characteristics, size, and other physical traits. Recognized genetic diseases in beagles include primary glaucoma, factor VII deficiency, and multiple epiphyseal dysplasia (Clark and Stainer 1994). Other health conditions that appear to be more common in laboratory beagles than other breeds are polyarteritis (beagle pain syndrome) and idiopathic epilepsy (BVAAWF/FRAME/RSPCA/UFAW 2004b).

Behavior is another critical characteristic in determining the quality of the research animal. An individual dog's response to stress and its propensity for developing abnormal behavior can be genetic (BVAAWF/FRAME/RSPCA/UFAW 2004b). For this reason, it is important for breeding facilities to ensure that they screen and cull dogs that have unacceptable genetic backrounds as well as unacceptable physical, physiological, and behavioral characteristics. Institutions can assist vendors by identifying dogs that have a genetic problem or condition with a suspected genetic etiology and communicating that information. This information is especially important for genetic conditions that may not be detected at an early age.

Early Behavioral Development and Socialization: Recommendations for Vendors

Dogs that are used for research purposes have the same requirements for socialization, foundation training, and early introduction to environmental and social stimuli as other working or pet dogs (Scott and Fuller 1965). To fulfill these requirements, breeders should follow guidelines such as those provided in Tables 1 and 2, which incorporate exposure to various novel situations and experiences during critical stages in a puppy's life and beyond. It is extremely important for their future well-being and adaptability to provide dogs with a variety of appropriate environmental, sensory, and social stimuli (Fox 1978; Fox and Stelzner 1966; Wolfle 1987).

Most Class A research dogs are raised in a breeding unit during their sensitive socialization periods and become adapted to these specific surroundings. They are usually transferred to another facility between 5 and 12 mo of age, and the new facility may differ greatly from the breeding unit (Boxall et al. 2004). If puppies and adolescent dogs have had limited experience to complex or novel situations, there most likely will be limits on their ability to adapt to subsequent environments and social complexities (Boxall et al. 2004). A scaling system for evaluating the socialization of puppies appears in Table 3. From the time dogs arrive and throughout their lives, facility personnel should handle them, play with them, and provide environmental diversification and trusting relationships.

Human Handling

Handling pups between birth and weaning has been reported to have effects on many characteristics, including rate of growth and weight gain, learning, exploratory behavior, emotionality, physiology, response to food and water deprivation, and the occurrence of some diseases or pathogens (NRC 1992). Early handling exercises in dogs have a lasting influence on the activity of the hypothalamic-pituitary-adrenal (HPA¹) system (reduced emotionality and increased stability) and may have a pronounced effect on the animal's basic temperament and future trainability (Lindsay 2000).

In many commercial breeding facilities where dogs are bred for placement at research facilities, puppies are housed in groups with limited time for human contact after weaning. For this reason, it is important for a program to be in place at the breeding facility to ensure that appropriate puppy-human interaction occurs during the critical period and beyond (NRC 1992). Handling of neonatal puppies for only 3 min a day and exposing the animals to various mild environmental stressors (e.g., changes of ambient temperature and movement, known as gentling) have had positive impacts on puppies' resistance to disease, emotional reactivity, mature learning, and problem-solving abilities (Lindsay 2000). Personnel should take into consideration the “critical period” of puppies to optimize their adaptation and to avoid their exposure to certain stimuli when they are most susceptible to developing problems.

Environmental Diversification

Breeding facilities should develop formalized programs that utilize knowledge of canine behavior. Programs should expose puppies to different noises, odors, enclosures, flooring or table surfaces, handling techniques, personnel, and restraint procedures. Careful environmental exposure carried out systematically through gradual increments of intensity and duration allows puppies to habituate to potentially fear-eliciting stimuli without undue distress. A puppy's environment should be rich in diversity of objects, textures, and structure with which to interact and explore (Lindsay 2000).

Trusting Relationships

An important part of training puppies is to give them confidence in people and to teach them that people have control by appropriately establishing humans as dominant figures in their social structure. Puppies learn to be subject to control from their mothers, and animal care staff must also teach them to accept human control and an understanding of hierarchical social structure. Developing trust in people helps puppies cope or adjust when new or scary situations are encountered and during handling for various procedures. Part of developing trust requires handling puppies appropriately and exposing them in a positive way to new and different individuals (e.g., different sexes, ages, ethnic backgrounds) and to potentially frightening situations (Wolfle 1987).

Breeders can develop simple or elaborate early canine socialization programs that can be successful. A critical aspect of any early socialization program is to establish a one-on-one relationship between each puppy and a person. An example of a puppy socialization program for dogs used in research is provided in Table 1 (BVAAWF/FRAME/RSPCA/UFAW 2004b). Suggestions for the social evaluation of puppies appear in Table 2 (Wolfle 1990).

Play

Play has many influential facets that profoundly affect developing puppies, especially with respect to adult social responsiveness and trainability (Horwitz et al. 2002; Lindsay 2000; McFarland 1981; Overall 1997). Play functions beneficially in the following ways: It stimulates communal behavior; facilitates social interaction, establishes strong social relationships, enhances physical and mental dexterity, improves coordination, and provides puppies with an outlet to learn about social rules. It also provides puppies with an outlet for exploration, and facilitates complex problem solving (Overall 1997). For these reasons, designated playtime should be part of any canine breeding program. Providing these basic experiences will help puppies develop coping skills for a wide array of situations, including those they may encounter at the research facility where they eventually will be housed. Breeders should continue acclimation and socialization reinforcement beyond the critical period (up to 6-8 mo of age) because dogs have been known to show behavioral regression when older (Boxall et al. 2004).

Transport to the Institution

Transportation, which is an especially powerful stressor for dogs, is a major life event for the animals, particularly when they are moved into new facilities and over long distances with multiple modes of transport (e.g., truck and air travel). Even moving within a facility can cause an animal stress (Swallow et al. 2005). During transport, animals experience a multitude of stressors, including the following: handling; a new enclosure; unfamiliar sounds, odors, and personnel; changes in feeding regimens; environmental variables; and separation from familiar conspecifics. Many reports of studies in dogs and other species have documented that the process of moving animals causes significant changes in the parameters used to assess stress, and that varying periods of time are required for values to return to baseline levels (Aguila et al. 1988 [mice]; Drozdowicz et al. 1990 [rodents]; Grandin 1997 [farm animals]; Kuhn et al. 1991 [dogs]; Landi et al. 1982 [mice]; Swallow et al. 2005 [laboratory animals]; Tuli et al. 1995 [rodents]).

One study evaluated 1-yr-old beagles transported for approximately 9.5 hr by truck to a new facility. Investigators found significant increases in plasma cortisol and corticosterone compared with nontransported controls, which returned to basal levels overnight after arrival (Kuhn et al. 1991). High cortisol levels have also been found in dogs transported within a facility when physiological parameters were compared in exercised and nonexercised dogs. The author (Clark 1997c) concluded that initially high cortisol levels were caused by the novel disturbance of transportation, relocation, and the new environment.

Thus, transportation can have a considerable impact both on canine welfare and on the scientific validity of any future studies involving the animals (Reilly 1998; Swallow et al. 2005). In an effort to minimize transport-related stress, the following approaches are recommended:

• Plan ahead;
• Follow appropriate regulations and guidelines;
• Have proper caging and facilities;
• Habituate dogs to transport caging before shipping;
• Examine and observe the dogs before, during, and after transit;
• Load the animals into cages and vehicles as close as possible to the time of departure;
• Ensure that personnel are knowledgeable and skilled in handling animals; and
• Follow proper procedures.

Animal welfare regulations and guidelines outline standards for transporting dogs (BVAAWF/FRAME/RSPCA/UFAW 2004b; CFR 1985; IATA 2000; Swallow et al. 2005). Keep in mind that the quality of the journey is more important than the duration with regard to minimizing stress (BVAAWF/FRAME/RSPCA/UFAW 2004b).

Newly Received Animals

It is essential to review the individual health records (e.g., vaccination history, diagnostic tests, clinical problems, treatments, surgical procedures) of each dog from the source facility soon after their arrival. It is optimal, when possible, to review the health histories before the animals are shipped, because this information will help in the selection of the most appropriate animals, will facilitate treatment of any pre-existing health problems, and will prevent animals from being shipped unnecessarily.

Stabilization

It is imperative to give newly received animals a period of time for physiological, psychological, and nutritional stabilization before their use. The length of time for stabilization will depend on the type and duration of animal transportation, the dogs involved, and the intended use of the animals (NRC 1996). A formalized program for quarantine and routine preventative medicine with written standard operating procedures is part of a high-quality colony management program. Good documentation systems should be in place, and detailed health records that meet current professional standards and regulatory requirements should be maintained (CFR 1978).

It is advisable to pair- or group-house dogs upon arrival and to subject them to a complete physical examination. Behavioral assessments should be conducted early in the quarantine period, and it is important to evaluate social structures when pair- or group-housing dogs (BVAAWF/FRAME/RSPCA/UFAW 2004b). Dogs' initial social encounters elicit stress responses, especially when they are introduced to unfamiliar conspecifics (Bayne et al. 2002); however, animals can adapt quickly if they are given the opportunity to work out dominance hierarchies appropriately (NCR 2003). One way to enhance acclimation to the new facility and avoid dog-to-dog aggression is for vendors to transport dogs to user premises in pre-existing stable groups (BVAAWF/FRAME/RSPCA/UFAW 2004b). Before moving dogs into group housing, animals can be acclimated through olfactory exposure (e.g., moving a toy with the other dog's scent between cages). A good predictor of aggressive encounters in dogs is body weight. Contrary to some conventional wisdom, it is best not to try and match cage-mates in size or weight. Matching canine cage-mates in size or weight may slow acclimation, prolong the period of hierarchy formation, and increase aggression (G. Fortier, Animal Behavior Institute, Furlong, PA, personal communication, 2006). Timid or fearful dogs should be identified early in the quarantine period, and a plan should be in place to address and reduce their stress.

Quarantine

Animals are particularly susceptible to the development of clinical problems from underlying pathogens and opportunists after undergoing the stress of transportation and being placed in a new environment (Swallow et al. 2005). Dogs may be asymptomatic carriers of pathogens (e.g., Campylobacter spp.) or have subclinical infections (e.g., giardiasis) before they are transported, and may later show signs of clinical disease upon arrival (BVAAWF/FRAME/RSPCA/UFAW 2004b, Chalker et al. 2003; Dysko et al. 2002, Greene 2000; Meunier et al. 2004; Tilley and Smith 2000). For these reasons, it is important for animals to undergo an adequate quarantine period (NRC 1996). Recommendations for canine quarantine procedures vary and are based on differences in facilities, source of animals, investigator requirements, and other issues.

Veterinary staff at each institution should use professional judgment in determining the duration and type of procedures dogs will undergo. Some institutions recommend canine quarantine periods of 7 to 14 days for newly purchased dogs, when minimal health and behavior problems are expected (BVAAWF/FRAME/RSPCA/UFAW 2004b). Quarantine periods may be lengthened when dogs have clinical problems, require additional diagnostic tests, have unknown health histories, need longer acclimation periods, and/or require treatment (BVAAWF/FRAME/RSPCA/UFAW 2004b; Hawkins et al. 2004). A program of veterinary care including diagnostic tests such as complete blood counts, clinical chemistries, fecal flotation, and cultures for enteric pathogens can help identify clinical or subclinical problems and facilitate timely treatment and preventative measures.

It is important for each facility to have an established process to acclimate animals into their new environment appropriately. Changes in routine quarantine procedures may be warranted, depending on the health status of the animals and study requirements. Having a formalized quarantine release process in place helps ensure that all aspects of the quarantine program have been completed. Documentation should include complete records, as mandated by GLP, when animals are placed on toxicology studies (CFR 1978, 1985).

Health and Preventative Programs

Routine Assessments

A process should be in place to ensure that complete and consistent health assessments are performed regularly to identify and address problems early. Many facilities perform monthly canine health assessments and routine preventative tasks that include the following brief examinations: assessing ears, eyes, oral health, haircoat, and skin; recording body weight and body condition score (BCS¹); cleaning ears; and clipping nails. Staff should be aware of any clinical problem that could affect a study. Complete physical examinations, vaccinations, dental prophylaxis, and other aspects of preventative medicine programs should also be performed, as deemed necessary, by the veterinary staff. Many institutions schedule annual veterinary examinations in conjunction with routine health maintenance programs (e.g., vaccinations, fecal parasite examinations, routine blood work).

Depending on the research goals, additional evaluations or diagnostic workups may be scheduled. Toxicology studies may require full veterinary examinations before studies. These examinations often include detailed ophthalmology examinations and full cardiac workups (e.g., ECG evaluations). Dogs that are immunosuppressed, have any surgical implant, or are being used for chronic studies may require additional preventative care and diagnostic procedures. For example, dogs with implanted devices (e.g., vascular access or intestinal access ports, telemetry units) should be clearly identified and the implants should be evaluated and/or maintained routinely (Adams 2002; Dysko et al. 2002).

Clinical Disease

Many research facilities have reported “interdigital” (ID¹) cysts in their dog colonies (Kovacs et al. 2005). The etiology of ID cysts (also known as interdigital pyoderma or canine pedal furunculosis) is often multifactorial (e.g., bacterial infections, mechanical, allergic). One hypothesis for the etiopathogenesis of sterile pedal furunculosis is an immune-mediated inflammatory response to keratin and triglycerides that are liberated from ruptured hair follicles, sebaceous glands, and the panniculus (Medleau and Hnilica 2006). Contributing factors include poor sanitation, wet environments, food allergies, mechanical injury (e.g., due to flooring), age of the dog, and BCS (Kovacs et al. 2005; Medleau and Hnilica 2006). At one facility, the incidence of ID cysts in a research colony of beagles was found to be 55% in ≥4-yr-old dogs. The type of raised flooring also contributed to the incidence of ID cysts. Data showed an incidence of 8% in diamond polyvinyl chloride (PVC¹)-coated floors, 26% on flat-bar steel (uncoated) floors, and 46% on flat-bar PVC-coated floors (Kovacs et al. 2005). At my institution, the concensus is that dogs housed in cages with flat-bar steel (uncoated) flooring have a higher incidence of ID cysts than dogs in kennels on solid flooring. It is important for facilities to institute appropriate husbandry practices to prevent and manage ID cysts in their dog colonies, along with appropriate treatment.

Mycoplasma haemoncanis (formerly Haemobartonella canis) was recently reported in dogs in a research facility in Germany. This red blood cell parasite is of particular concern in kennel-raised dogs that are immunocompromised. Occurrence of clinical disease caused by M. haemoncanis resulted in failure of a large experimental project in splenectomized dogs in a hemorrhagic shock study (Kemming 2004a,b). This report raises concerns about the potential for latent infections to adversely affect or confound research, especially in immunocompromised models (e.g., dogs used in transplantation studies).

Vaccinations

Vendors and research facility personnel are responsible for vaccinating dogs according to recommended professional guidelines and regulatory requirements (e.g., state and local laws). Core vaccinations should be included in any quality preventative programs unless doing so would interfere with studies. Core vaccines are defined as vaccines appropriate to provide protection in most animals against diseases that pose a risk of severe disease because the pathogens are virulent, highly infectious, and widely distributed in particular regions (Klingborg et al. 2002). Generally, core canine vaccinations include canine distemper, canine parvovirus (CPV¹), hepatitis, and rabies. Additionally, noncore vaccinations should be given as determined by exposure risk factors (Paul et al. 2003).

Recently, recommendations have changed regarding the intervals for vaccinating dogs because of updated information on duration of immunity, risk factors, and improvements in the field of vaccinology (Hendrick and Dunagan 1991; Paul et al. 2003; Shoenfeld and Aron-Maor 2000). Previously, routine annual vaccinations were recommended for most dogs after 1 yr of age. However, new information regarding duration of immunity now supports lengthening the frequency between vaccinations with core vaccines (Mouzin et al. 2004; Paul et al. 2006; Schultz 2000). Many veterinarians currently support triennial vaccination against core diseases following initial booster revaccination at 1 yr of age with the appropriate vaccines (Greene et al. 2001; Paul et al. 2003). These new recommendations may assist in decreasing vaccination and study scheduling conflicts as well as preventing the incidence of adverse vaccine-induced side effects.

Knowledge of a dog's previous history and potential exposure helps in identifying potential risk factors and the type of vaccination and preventative medicine program that should be administered in each individual case. Facilities that purchase random-source dogs may choose to vaccinate their dog colony with noncore as well as core vaccines, because dogs may have questionable vaccination histories and greater potential for exposure to or transmission of infectious diseases. Institutions that purchase purpose-bred dogs may choose to set up a vaccination program with only core vaccines.

In some situations (e.g., canine vaccine studies), it may be necessary to not administer all or some recommended vaccinations and additional precautions to ensure that the dogs are not exposed to potential pathogens. For animals designated for vaccine studies, stricter barrier procedures should be implemented (e.g., use of more stringent personal protective equipment and barrier entry practices).

If dogs require vaccinations, investigators and veterinary staff should always be aware of potential effects to the study or in scheduling studies. Vaccinations stimulate the immune system and occasionally result in vaccine-related adverse effects (Hendrick and Dunagan 1991; Shoenfeld and Aron-Maor 2000). For example, transient lymphopenia occurs 4 to 6 days after administration of some attenuated CPV vaccines (e.g., CPV-2); humoral immune responses are similar to those observed with field CPV (Hoskins 2000). Questions have also been posed regarding a connection between vaccination and autoimmunity (Shoenfeld and Aron-Maor 2000). A study in puppies immunized with a variety of commonly given vaccines showed a possible causal relation between vaccines and autoimmune findings (Hogenesch et al. 1999). For these reasons, investigators who place dogs on studies involving immunity should be aware of the potential associations between vaccinations and immune responses and how they could affect a study. To minimize vaccination as a variable, dogs should be vaccinated no later than 2 to 3 wk before they are placed on study (D. Herzyk-Hrebien, GlaxoSmithKline Pharmaceuticals, personal communication, 2006).

Dental Prophylaxis

Oral health is an important part of a dog's good general health and acceptability for study. Gingivitis is reported in ≥70% of dogs by 2 yr of age, and most dogs have some degree of periodontitis by the time they are 5 yr old (Harvey 1998; Wiggs and Lobprise 1997). Because periodontal disease can have a significant impact on animal health and may complicate studies, it is imperative to perform regular oral evaluations to ensure dogs' good dental health (DeBowes 2000; DeBowes et al. 1996). Methods for evaluating the severity of gingivitis and accumulation of dental deposits have been developed using scoring systems for humans that have been validated for use in animals (Gorrel and Bierer 1999; Harvey and Emily 1993; Hefferren et al. 1994; Loe 1967; Logan and Boyce 1994; Wiggs and Lobprise 1997). The reader is referred to the literature on recommended dental treatment and preventative protocols (DeBowes 2000; DuPont 1998; Holmstrom et al. 2004) as well as standards for dental diets published by the Veterinary Oral Health Council (VOHC¹) of the American Veterinary Dental College (www.vohc.org).

Exercise

Exercise is essential for dogs. In the United States, animal welfare regulations (CFR 1985) require giving dogs the opportunity for exercise. Each institution is responsible for developing a plan for providing canine exercise with the approval of the attending veterinarian. When doing so, staff should consider the types of research protocols to which dogs may be assigned and any current or potential health-related issues of concern. Exercise and procedures associated with an exercise program (e.g., handling, transport, and socialization with conspecifics and humans) may also affect study results (BVAAWF/FRAME/RSPCA/UFAW 2004b; Campbell et al. 1988; Hetts et al. 1992, Hubrecht et al. 1992; Hughes and Campbell 1990; Raekallio et al. 2005). A consistent exercise program should be in place for all dogs on a particular study, taking into consideration that there will be some individual animal and situational differences. For instance, it may not be possible to exercise all dogs on a single study at the same time due to sex differences, space constraints, and animal compatibility.

If dogs cannot be exercised according to an institution's exercise plan for scientific reasons, then the IACUC must approve the written exemption. It is important to appropriately sanitize exercise areas between groups of dogs, and to exercise together only dogs of similar health status. Staff should be encouraged to interact and play with the dogs during exercise periods (Campbell et al. 1988; Hughes and Campbell 1990). It is preferable to have same-sex exercise groups, especially if dogs are intact. It may be necessary to take special precautions with females if they are in estrus, due to heightened sensory stimulation and potential aggression.

Enrichment

The importance of providing dogs with sources of enrichment is well recognized. Primary sources of enrichment include interaction with people and the provision of manipulanda, as described below. Two useful resources regarding enrichment strategies for laboratory dogs can be found in a recent article by Overall and Dyer (2005) and the publication by the BVAAWR/FRAME/RSPCA/UFAW Joint Working Group on Refinement (2004b). A list of enrichment recommendations can be found in Table 4.

The single most important enrichment modification for dogs is to have humans handle them frequently and in a wide variety of circumstances (Overall and Dyer 2005). Personnel can take initiatives to interact with the dogs during routine facility walk-throughs, when checking water lines, during exercise and acclimation sessions, and during routine health assessments. To ensure canine-human interaction, institutions should allow adequate time for personnel to fit this activity into their routine daily schedules.

Toys can also provide a form of positive stimulus for dogs. A wide assortment of canine toys is available and can afford novelty and variety as part of a canine colony enrichment program. Manipulata should be evaluated and utilized based on agreed-upon criteria such as the following: extent of use by and benefit for the dog, durability, no or minimal study impact, ease of sanitation, potential to transmit disease, animal and personnel safety, minimal risk of adverse effects (e.g., clogging drains), resources involved, and cost.

Evaluation of toy use may be difficult, particularly when one or more toys are placed routinely in the dog's cage or kennel. In my experience, dogs focus on staff and usually are not seen playing with toys when personnel are present in the room. Often, the only way to assess use is by physical evidence (e.g., chew marks) or by video recording the dog's activity. At our facility we have also noticed that during group exercise sessions, dogs play more readily with soft toys (e.g., sheepskin frizzbies) or with tug toys. These toys also appear to decrease dog-to-dog aggressive and sexual behaviors. Dumbbell-like toys are frequently used by single or paired dogs at GlaxoSmithKline Pharmaceuticals.

Nutrition, Feeding Regimens, and Weight Management

A dog may not adapt readily to dietary and environmental changes, so close attention should be paid to be certain that a newly arrived animal is eating adequately and maintaining its weight. Optimal body weight and body condition are very important both for the health of the animal and to ensure good study results (Burkholder 2000).

The following characteristics of each dog must be taken into account when managing its nutritional requirements in a laboratory setting: age, sex, breed, metabolic rate, size, activity, body weight (BW¹), body mass index, body composition (e.g., amount of body fat), BCS, and eating habits. Significant breed differences can be seen in the feeding behavior of dogs. For example, it has been reported that beagles have feeding patterns similar to cats when fed free-choice (e.g., eating 10-20 times/day between the light and dark period). Adult dogs do adapt to one meal/day feeding (Morris et al. 2005). Other factors to consider in diet management include type, duration, and schedule of the study to which the animal will be assigned; study parameters; route of drug administration; need for fasting the animal; the animal's potential for placement on multiple studies; and its longevity in the colony.

Body Condition Score

A BCS provides a perspective for the amount of weight an animal carries relative to its skeletal frame (i.e., body fat: nonfat tissues ratio). BCSs have been used in clinical assessment for determining optimal nutrition (Burkholder 2000; Laflamme 1997). Assessing body condition by assigning a BCS is a subjective, semiquantitive method of evaluating body fat and muscle. Although body condition may sometimes be correlated with body weight, a BCS is generally independent of weight or frame size.

Various scoring systems have been devised to evaluate body fat and muscle. The scores correlate visual and palpable measurements for a particular region of the animal (e.g., ribs, tail base, and abdomen) to an assigned number (generally using a 5-, 6-, or 9-integer scale). A 5-point system is most useful to novices, and a 9-point system is used more frequently by experienced clinicians (Remillard 2005). Use of body condition scoring is useful in assessing whether animals are thin, normal, or overweight and whether dogs should be fed more or less (Burkholder 2000). A dog in optimal body condition (e.g., BCS of 3:5 or 5:9) has “normal body contours and silhouettes, bony prominences that can be readily palpated but not seen or felt above skin surfaces, and intraabdominal fat insufficient to obscure or interfere with abdominal palpation” (Remillard 2005, p. 76). Dogs with an optimal dog body condition have 15% to 25% body fat (Remillard 2005). Dogs with ideal BCSs live 15% longer and have a decreased prevalence of degenerative orthopedic disorders and some cancers (e.g., mammary tumors, cystic transitional cell carcinoma) (Remillard 2005).

Maintenance Energy

Maintenance energy (ME¹) requirements of adult dogs may be estimated from formulas relating ME requirements to BW or resting energy requirements. Formulas are a useful starting point but are not absolute requirements because there is considerable individual animal variation (Butterwick and Hawthorne 1998; Heusner 1991; Morris and Rogers 2000; Richardson and Zicker 2000). Meal feeding with an optimal amount of feed (food optimization), rather than ad libitum feeding, should be the norm for all dogs (Fillman-Holliday and Landi 2002). Adult dogs that are consuming food voluntarily should be fed relative to their body condition and not by a formula (Morris and Rogers 2000).

Feeding Regimens

Feeding regimens at institutions vary and are dependent on study requirements. There are basically three methods of feeding dogs: free choice (ad libitum), time limited, or food limited. In many canine research colonies, dogs are fed a set amount of food based on an average size, frame, and/or weight of the dogs in the colony. Staff may feed a set amount to ensure that no dog is below or above a set body condition or because of the convenience of not having to calculate individual feeding regimes. It is preferable for feeding regimens to be individualized, but that preference is not usually feasible or necessary. Feed amounts can be calculated based on BCS, frame, age, and/or sex. For example, the amount of food given to male dogs may be greater based on a larger average frame and acceptable BW and BCS. It is advisable to weigh each dog and to assess its BCSs monthly. It is important to monitor the monthly BCSs for changes and to address changes as needed.

It may be necessary to separate pair- or group-housed dogs during feeding because of individual differences in ME requirements, variations in feed consumption, and social dynamics (e.g., dominance) that can occur over food. If it is necessary to place a dog on a weight reduction program, staff should ensure that appropriate feeding regimens and/or diets are used. A dog can be placed on a weight loss program by being fed its regular food reduced by ≤25%. If it is necessary to decrease the amount of a dog's feed by >25%, staff should feed an approved diet specified for weight loss; otherwise nutritional deficiencies may occur (Remillard 2005).

Toxicology and safety pharmacology studies have particular regulatory requirements that often affect decisions related to nutritional management and feeding regimes. Study directors typically feed the same certified diet and standard amount of food to all dogs on a study to minimize variables. Dogs should be placed on study diets well in advance of starting the study (Fillman-Holliday and Landi 2002). Investigators may choose to have limited scheduled feedings (i.e., food available to the animals for a set amount of time at the same time each day, and then removed) to facilitate fasting, dosing, and sampling of the animals. When feeding is limited for shorter periods, some dogs focus their attention on personnel and/or activities being performed in the room, which can decrease food consumption. For this reason, dogs may need a quiet period during limited feeding times to focus on eating the food offered them. The change to a new feeding schedule should also be gradual—over several days—to ensure that the dogs have become accustomed to the new schedule before the start of a study.

Understanding the Role of the Stress Response as an Experimental Variable

Biology of Stress

“Stress is the effect produced by external (i.e., physical or environmental) events or internal (i.e., physiologic or psychologic) factors, referred to as stressors, which induce an alteration in an animal's biological equilibrium” (NRC 1992, p. 3). Stress can result when an internal need or environmental demand is perceived by an individual as threatening or out of its control (Clark et al. 1997a). An animal's response to stress can result in elevations in corticosteroid levels, along with other physiological changes, and may be characterized by concurrent behavioral changes (Hetts 1991). Stress biology involves a complexity of general and integrative responses by the central nervous system, autonomic nervous system, HPA axis, and target organs, which together induce changes in the homeostatic processes directed toward maintaining an animal's physiological equilibrium. These responses affect a dog's physical and psychological state, behavior, immune function, and morphology (Clark et al. 1997a).

The HPA axis is particularly sensitive to psychogenic stressors such as novel or threatening surroundings, separation from attachment objects, unpredictability of external events, or lack or loss of control over environmental contingencies (Hennessy et al. 1998). Whether stress will lead to distress with the onset of maladaptive behavior and physiological and pathological changes in the animal depends on the intensity and duration of the stress and the animal's adaptability (NRC 1992). When a dog adapts positively to stress, it successfully modifies its behavior, metabolism, and physiology with an outcome that is positive for the animal. If the dog is unsuccessful in developing adequate responses to enable it to cope, the response can then become maladaptive and the dog can develop subsequent affective disorders, disabilities, dysfunctions, and/or diseases. The subsequent developments can have significant study implications (Clark 1997a; Shull-Selcer and Stagg 1991).

Fear and Anxiety

Fear and anxiety are two important emotional responses that can occur when animals undergo experimental stressors. These responses are part of an animal's normal behavioral repertoire to help protect it from harm.

Common procedures and situations that can elicit fear or anxiety in laboratory dogs include cage changing, removal from a stable social group, modification of established maintenance routines, transportation, confinement in a strange setting, restraint, procedural manipulations (e.g., injections, dosing, sampling techniques), wearing equipment (e.g., jackets), introduction to new people or conspecifics, and association with a previous negative experience (Beerda et al. 1999; BVAAWF/FRAME/RSPCA/UFAW 2001, 2003, 2004b; CCAC 1993; NRC 1992). The addition of novelty to a potentially stressful experience increases the intensity of an individual stressor (NRC 1992). To assess the impact of aversive stimuli in a research setting, it is necessary to consider factors such as the intensity, quality, frequency, duration, and rate of change of the stimulus and whether it is novel, incidental, or routine (Clark 1997b).

Physiological Data and Stressors

Stress responses may cause variability in studies for which pharmacological or physiological data are gathered (Overall and Dyer 2005). In experimental procedures that assess physiological measurements such as heart rate (HR¹), blood pressure (BP¹), and ECGs, stressors can significantly affect data (Adams 2002). To minimize these potential effects, it may be necessary to implement longer and more intense acclimation and training.

Restraint is known to have effects on hemograms, plasma hormones, and other parameters in dogs, monkeys, and other species (Clark 1997c; Landi et al. 1990; Slaughter et al. 2002). In dogs, increases in catecholamines due to stress can induce pronounced splenic contraction and substantial increases in blood volume due to the relatively large canine splenic capacity (Reeve et al. 1953). This effect can cause changes in hematological, clinical chemistry, and physiological parameters (Joles et al. 1982; Sato et al. 1995). In a study in which the effects of periodic blood sampling on vasoactive hormones and blood volume in dogs were evaluated, investigators concluded that epinephrine-induced splenic contraction, initiating from the stress of blood sampling, caused higher hematocrit values during 14 days of study (Slaughter et al. 2002).

Acclimation and Training Programs

The goals of an effective acclimation program are to ensure that animals are well adapted to the potential experiences and social interactions to which they will be exposed, thus enhancing the animals' well-being and minimizing experimental variability. Attaining these goals can be challenging due to a multitude of potential environmental, social, and procedural stressors as well as individual animal variation (CCAC 1993). Because of stress response variability between individual animals, the degree of acclimation may vary, and it may be difficult to anticipate stress-related changes in specific parameters (Gaebelein et al. 1977; Galosy et al. 1979). In general, novelty, the absence of predictability, and the lack or loss of control cause an increase in the activity of stress-responsive physiological systems (NRC 1992). Decisions related to the intensity or time involved for acclimating dogs can be based on previous knowledge, breed differences in behavioral trends, experience, and close observations of the individual animal before and during exposure to a procedure or stimulus (Hetts 1991).

Role of Facility Personnel

As described above, it is best to have formalized acclimation, socialization, and training plans in place before dogs arrive at a research facility. Animal care staff must be familiar with experimental requirements and have a plan in place to facilitate the process used to ensure that dogs meet all of the study criteria in advance of experimental procedures. Personnel who perform acclimation procedures should (1) have a thorough knowledge of species-specific and breed-specific behaviors, (2) be familiar with the individual animal's temperament, (3) be aware of situations and procedures that animals under their care will likely experience, (4) understand the potential risk factors for the animal and personnel involved in the acclimation process, and (5) use appropriate acclimation and training techniques.

Personnel who utilize the following recommendations will facilitate animal acclimation and promote the successful use of animals in studies:

1. Allow adequate time to closely monitor and interact with new arrivals. Many dogs will be timid upon arrival and exhibit fear or anxious behavior. Dogs should be encouraged to approach staff through positive interactions—petting and appropriate verbal communication.
2. Spend time with new dogs. This investment can significantly improve how well the animals adapt to their new environment and will most likely decrease the time needed to acclimate appropriately (see Early Behavioral Development and Socialization; Table 1).
3. Perform individualized behavioral assessments on each dog to help identify behavioral issues (Boxall et al. 2004). Assessments can be used to assist in addressing behavior problems and in assessing an animal's individual behavior before its placement on study.
4. Give dogs adequate time to recover from transportation stress and to adapt to their new facility (see Newly Received Animals). The recommended acclimatization period is at least 7 days after transport that has involved a journey in a vehicle and between sites, and at least 3 days when they have been moved in individual containers between buildings on-site (BVAAWF/FRAME/RSPCA/UFAW 2004b). At least 24 hr should be permitted for acclimatization when there is a permanent change of pen location, assuming similar husbandry and care procedures are followed (BVAAWF/FRAME/RSPCA/UFAW 2004b).

Daily Situations

Adapting dogs to daily situations like cage changing, feeding regimens, exercise, and socialization with humans and other dogs should be incorporated into daily and weekly routines (CCAC 1993; Turner et al. 2003). The following additional guidelines are recommended:

1. Allow extra time to observe, interact with, and handle newly arrived dogs during routine procedures like cage changing and water checks.
2. Provide a diet that is the same as that of the kennel of origin, if possible. The diet can then be changed over several days to a new food if a diet change is necessary.
3. Pair- or group-house compatible dogs because social interactions help minimize dogs' fearful and aggressive behavior (Adams et al. 2004; Hubrecht 1995; Overall and Dyer 2005; Wolfle 1990). Placing together dogs that have been previously housed in compatible groups is preferable although often, newly arrived dogs have been separated from known cage mates.
4. Set aside time with each individual dog to help with the animal's transition into the new facility and facilitate formation of new social attachments with unfamiliar humans and conspecifics.
5. Give dogs the opportunity to smell their surroundings and perform canine introductory rituals. Dogs utilize their keen sense as a means of social communication and allowing them the opportunity for olfactory experiences helps to enhance their acclimation to new environments.
6. Follow planned husbandry and care procedures while being aware of each individual dog's behavioral characteristics and adjust procedures if any dog shows signs of heightened anxiousness or fear. When an animal or a group of animals shows a strong motivation to avoid or escape a stimulus, the situation warrants additional attention or intervention. Some dogs may need to avoid environments that are too challenging for them at a particular time (Overall and Dyer 2005).
7. Acclimate and train dogs appropriately, well in advance of the time they will be on study.
8. Performance of common restraint and health-related procedures such as physical examinations, ear cleanings, and nail trimming can help prepare dogs for other procedures performed while on study (e.g., blood collection, placement of intravenous catheters, injections, oral dosing, and placement in slings).
9. Handle and manipulate the dogs in a manner that is similar to the handling of procedures when the animal is on study.
10. Train the dogs so that they are familiar with the transport cages that will be used for their relocation to laboratories or testing areas well in advance of the study. If other dogs will be transferred with an animal, appropriate socialization with these animals should be part of the acclimation process.

It may be helpful to set up planned situations to expose dogs to aversive events. After exposure to aversive events under highly controlled and predictable conditions, dogs appear to learn how to cope more competently with a subsequent aversive situation (i.e., to show less distress and physiological arousal than when such events occur uncontrollably). As a dog's competency and confidence improve, the animal becomes more relaxed. Relaxation competes with fear and anxiety (Lindsay 2005).

A phased acclimation plan is useful when experiments involve multiple procedures. For example, personnel can first remove the dog to a novel environment for an adequate acclimation phase, next acclimate the animal to sling restraint, and subsequently acclimate the dog to oral dosing. Animals habituate to one or more procedures best when the equipment and handling procedure are species appropriate, sized correctly for the animal, and otherwise adjusted to maximize the animal's comfort (NCR 2003).

It is also important for dogs to become familiarized with key personnel who will be involved in an experiment, and for the animals to have developed some level of trust with each person. The goal of a good acclimation program is to minimize the stress response of the dog while exposing it to new situations and/or aversive stimuli and still have the animal perceive the experience as positive.

Basic Handling

Many references on training can be useful in developing basic handling and restraint techniques (Colflesh 2004; Donaldson 1996; Marder and Reid 1996; Pryor 1999). Basic training facilitates a dog's ability to control significant events competently, thereby promoting expectancies that are conducive to enhanced confidence, relaxation, and feelings of safety (Lindsay 2005). Positive interactions and reinforcement methods are the preferred ways to train animals (BVAAWF/FRAME/RSPCA/UFAW 2001, 2004b; Laule 2003; Turner et al. 2003). Using appropriate communication (e.g., tone and mannerisms) decreases a dog's anxiety and physiological variability (Wolfle 1990). Placing an animal in familiar surroundings helps decrease fear or anxious responses. For example, when teaching puppies restraint techniques, it is best to do so in familiar surrounding.

Stroking and handling by humans can be a practical and effective technique for calming animals in situations where they are distressed, particularly animals that have been positively socialized by humans. Early studies have shown that petting a dog can have a calming effect on the animal (e.g., decreases heart and respiratory rates) (Gantt et al. 1966). Petting may be an effective way of reducing canine cortisol responses to aversive situations such as physical examinations, injections, and blood sampling (Hennessy et al. 1998). The effect of stroking a dog has also been shown to increase affiliative neurochemicals in both dogs and humans (Odendall and Meintjes 2003).

Massage and relaxation training techniques have many applications in the management of dog behavior, especially in situations involving aversive emotional arousal (Hennessy et al. 1998; Lindsay 2000). However, care should be taken not to unintentionally reinforce fearful responses by overpetting the dog (BVAAWF/FRAME/RSPCA/UFAW 2004b). For example, vocal encouragement and petting can have a calming and beneficial effect on a moderately fearful dog but may inadvertently reinforce fearful behavior by providing the dog with a shield of safety behind which it can escape or avoid fearful situations (Lindsay 2005). The procedure should be used carefully with animals that are fearful or that might transmit disease (NRC 1992).

Investigators should consider which different handling styles may affect physiological and behavioral responses, especially in studies for which pharmacological or physiological outcomes are an important part of the data (Overall and Dyer 2005). For these types of studies at our institution, habituation to equipment, personnel, and other stressors usually requires ≥4 wk of training at least two to three times each week to ensure that animals are adequately acclimated and ready for study.

Restraint

Adaptation to restraint is especially important before animals are placed on experimental protocols that require restraint. Such adaptation is particularly relevant if dogs are to be restrained frequently or for long periods, or if the restraint method is especially rigorous (NRC 1992, 1994, 2003).

“Physical restraint is the use of manual or mechanical means to limit some or all of an animal's normal movement for the purpose of examination, collection of samples, drug administration, therapy, or experimental manipulation” (NRC 1996, p. 11). Canine habituation and adaptation to experimental procedures can have an impact on whether an animal will become distressed during experimental procedures. For example, animals that have habituated adequately to noninvasive procedures such as electroencephalogram and ECG measurements would not be expected to experience distress during data collection, whereas animals not adequately habituated would be expected to experience minimal distress initially (NRC 1992). The animals' distress, however, could be alleviated later during the study with the use of appropriate acclimation procedures and handling techniques. Habituating dogs to procedures associated with blood sampling also can be helpful in decreasing variability for certain research projects.

Restraint can involve restricting movement, positioning an animal by physical means, or placing the animal in some type of device (e.g., restrainer or sling). Animals habituate to the procedure and device better if the equipment and handling procedure are species appropriate, correctly sized for the animal, and otherwise adjusted to maximize the animal's comfort (NRC 2003). Body slings are used frequently to support dogs for prolonged periods in particular studies (e.g., serial sampling for pharmacokinetic studies, inhalation studies). Introduction to slings can be usefully incorporated into the initial habituation process in the breeding establishments, but specific habituation to the sensation of being suspended by these devices should also be provided before the individual studies begin, if the devices will be required (Turner et al. 2003). Dogs restrained in slings should be monitored at all times.

When selecting dogs for protocols that require restraint, it is important to screen each dog to ensure that the animal is in good health and has performed well during behavioral assessments. Most acceptable dogs, when scored from 5 (best) to 1 (least) on the following seven parameters, receive a positive average score ≥4.0: (1) General behavioral appearance with the cage/run door closed, (2) response after opening the cage door, (3) response to different clothing, (4) response to handling, (5) reaction to restraint, (6) response to being on the floor, and (7) response to having minimal restraint. Documenting specific comments about each dog will assist investigators' in future selections for specific studies or restraint devices.

The following pretraining steps will set the stage for a positive experience before the dog is placed in or on a restraint device:

1. Include the individual(s) who train the dog in the process;
2. Bring each dog to the training area for 5-10 min by itself;
3. Place each dog on the sling base (for slings) or training table (for boards);
4. Handle each dog calmly while manipulating its ears and legs and opening its mouth;
5. Offer treats; and
6. Allow each dog to explore the room at the end of the session.

After a dog has successfully completed the pretraining, it is imperative that the animal is given adequate time to adapt to restraint before being placed on experimental protocols that require restraint. Recommended guidelines and helpful hints for acclimating dogs to slings and boards appear in Table 5. Such adaptation is particularly relevant if dogs are to be restrained frequently or for long periods, or if the restraint method is especially rigorous (NRC 1992, 1994, 2003).

Animals fitted with jackets or equipment that delivers inhalation drugs require special, additional adaptation. It is particularly useful to work with breeders to identify very cooperative dogs before acquiring the animals for inhalation studies in which face masks will be utilized for inhalation administration of compounds (S. Goelet, Charles River Laboratories, Senneville, Canada, personal communication, 2004). When dogs are required to wear jackets, staff should ensure that the animal can tolerate the equipment. Some facilities have required up to 1 mo of frequent habituation to jackets before placing animals on study (BVAAWF/FRAME/RSPCA/UFAW 2003). At GlaxoSmithKline, we have found that 2 to 3 mo of training are required for jacket and tether training for 7- to 8-hr and 24- to 48-hr time periods, respectively (G. Rivera, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA, personal communication, 2006).

Some studies require dogs to lie in a particular position on a table or board for sampling or diagnostic tests (e.g., collection of bile via access port). Board training has been useful for teaching dogs to lie still during continuous urine collection and for preparation for ultrasound imaging. Techniques such as “dominance down” have been useful at our facility in habituating dogs to lie in a prone position (J. Dugan, SmithKline Beecham, personal communication, 1996) The process involves teaching the dog to accept lying in this position using positive reinforcement.

Using Behavioral Modification to Enhance Canine Acclimation and Training

Acclimation and training programs for dogs used in research have been developed with the following goals: to enhance the welfare of the animal, to decrease study variability, to ensure the safety of the animal and personnel, and when possible to minimize the use of resources. Cynopraxis is the term used to describe a pragmatic process of training dogs that enhances the human-dog bond while improving the dog's quality of life. There is now extensive literature regarding the theory, philosophy, and ethics of cynopraxic training, which can be helpful to staff who care for and train dogs (Lindsay 2005).

It is always preferable to train laboratory animals to cooperate during procedures and husbandry, rather than to physically restrain them, unless there is likelihood that they will injure themselves or staff. Such training helps reduce the stress associated with procedures and is an essential part of everyday husbandry (Turner et al. 2003). Attention to subtle procedural details and stimuli not only adds to the understanding of animal well-being but also reduces variability in data and facilitates comparison among closely related studies (Clarke et al. 1997b). Training with positive reinforcement is also useful as mental stimulation and behavioral enrichment (Overall and Dyer 2005).

All facility personnel who work with and care for puppies, adolescent and adult dogs can help the animals adapt to and cope with stressors by using various techniques such as acclimation, adaptation, and coping (Bayne 2002; BVAAWF/FRAME/RSPCA/UFAW 2001; Table 2). A variety of additional forms of behavioral modification and learning paradigms such as habituation, desensitization, and counterconditioning have been useful when animals exhibit fearful or anxious responses (Table 2).

There is a wealth of information on behavioral management and learning paradigms to help staff become more knowledgeable and proficient at using appropriate training techniques (Horwitz et al. 2002; Lindsay 2001, 2005; Overall 1997; Pryor 1999). Facilities can also consult with animal behaviorists to enhance their training programs and to problem-solve when challenging behavior issues arise.

Choosing the Appropriate Dog for the Experiment

As described above, some dogs have genetic predispositions for undesirable temperaments and physical problems (Overall 2000; van den Berg et al. 2003) (see Vendor Health and Genetic Considerations). It is advisable for investigators and veterinary staff to compile a list of acceptable (or unacceptable) physical, behavioral, and physiological criteria for dogs in particular research protocols (BVAAWF/FRAME/RSPCA/UFAW 2001, 2003; NRC 1992) and to communicate this information to breeders or vendors before dogs are ordered.

If a dog has a significant underlying behavioral abnormality that is genetically based or attributable to significant molecular or neurochemical abnormalities, then it will be more difficult to address any related problem. Breeders should screen out puppies and parents identified with genetic predispositions or behavioral characteristic that may be detrimental. However, it is not always possible to detect behavioral abnormalities at a dog's early age because some genetically based behavioral abnormalities are not apparent until a dog is older (Overall 2000). When making decisions regarding the placement of dogs on various studies, personnel should consider well in advance any special health or behavioral concerns that should be addressed before study. Dogs with chronic health problems (e.g., ear infections, dermatitis, interdigital cysts, diarrhea) are not the best candidates for long-term studies or studies involving immunosuppression or implanted devices (BVAAWF/FRAME/RSPCA/UFAW 2004a). If possible, it is advantageous for investigators to have stock dogs available that have already had some acclimation and training for common experimental procedures. These dogs can also be screened to identify any behavioral characteristics that would make them more or less suitable for certain studies.

Special Study Considerations

Dogs are often used in studies that involve dosing and/or sampling procedures, which sometimes include frequent and repeated sample collection. For such studies, placing intravenous (IV¹) catheters or implantation of access ports can facilitate blood sampling, dosing, and/or data collection (Adams 2002). It is advisable to follow standard guidelines for placement of intravenous catheters and for ensuring that exteriorized catheters are secured in place before utilization. Dogs used on chronic studies that require serial blood sampling, bile sampling, or blood pressure measurements can be surgically implanted with an access port placed in a vein, artery, or bile duct. Access ports are surgically implanted internal devices that have the advantage of repeated use for long periods and reduced likelihood of infection compared with exteriorized catheters (Kissinger et al. 1998; Mann et al. 1987). The ports have a dome that is sutured in place in an accessible subcutaneous site with an attached catheter secured in a vessel or duct. The femoral vein and artery are frequent implantation sites for vascular access ports in dogs, but other vessels such as the jugular and portal veins are also used. Care must be taken to appropriately maintain these devices, particularly with regard to preventing infection. To facilitate catheter or port patency, a heparin-saline or heparin-glucose lock can be used (Kwei et al. 1995).

In some situations, investigators may choose to place drugs directly into different segments of the gastrointestinal tract. Numerous procedures have been used to facilitate direct gastrointestinal dosing including implantation of biomedical devices and surgical manipulation of the intestines. Access ports are sometimes implanted into the gastrointestinal tract of dogs for long-term dosing (Meunier et al. 1993).

Radiotelemetry is a method used for capturing physiological parameters such as body temperature, HR, ECG, and BP in addition to movement, electromyography, and electroencephalography (BVAAWF/FRAME/RSPCA/UFAW 2003, 2004a). Telemetry offers investigators the opportunity for continuous monitoring while dogs are in their home cage. These devices can be implanted in various sites. Because stress responses can affect particular studies in which physiological measurements such as HR and BP are being taken, dogs should be carefully screened, and only those animals considered well adapted to a variety of situations should be chosen for these types of studies (BVAAWF/FRAME/RSPCA/UFAW 2004a).

Pain can trigger significant stress responses and result in study variability. Veterinary care staff must incorporate effective pain management practices for procedures that involve painful protocols (CFR 1985; NIH 2002; NRC 1992, 1996). A good pain management program must include pre-emptive analgesics; use of the most appropriate analgesics; and close, consistent, and frequent patient monitoring. For animals undergoing surgical procedures, appropriate surgical practices, postoperative care, and analgesics must be provided to facilitate recovery and to increase the likelihood that the animals will be in good health and ready for study.

Good pain management programs significantly enhance animal well-being and facilitate having a quality animal ready for study in a timely manner (AAHA 2003; BVAAWF/FRAME/RSPCA/UFAW 2003; Stasiak et al. 2003).

Animals may require several weeks of recovery after surgery, depending on the procedure. Importantly, all of the information related to pain management must be provided in detail in the IACUC-approved protocol.

Staff Training

Behavioral management is an important training aspect for all personnel working with dogs. Online resources as well as printed, video, and CD/DVD materials are now available through various institutions, organizations, and universities. Additional resources include the American Association of Laboratory Animal Science (www.AALAS.org) and its affiliates such as Laboratory Animal Welfare Training Exchange (www.lawte.org) and the Institute for Laboratory Animal Research (www.national-acadmies.org/ilar) (NRC 1991; www.lawte.org, www.aalas.org).

Conclusion

Preparing dogs for experimental procedures is a comprehensive process that starts at the breeding facility and continues throughout the lives of the dogs. Facilities should ensure that breeders, laboratory care staff, and researchers work together to optimize the chances of using the most appropriate dogs for research. Care staff must take into consideration the physical as well as the behavioral characteristics of a dog to maximize the well-being of the animal and to help minimize study variability.

Preventing and addressing health problems promptly helps ensure that dogs will be in good condition for study. Behavioral management is a very important aspect of decreasing stress and distress and thus minimizing the potential for study variability. For these reasons, administrators and staff should ensure that a high-quality behavioral management program is given a high priority at their institution. Adequate resources and well-trained staff who are knowledgeable and available to oversee canine colony management and care are important components of any high-quality animal care program. Effective planning and communication between care staff, investigators, and IACUC members are also key factors in preparation for all studies.

It is hoped that the information and recommendations in this article will be useful not only for helping to enhance the well-being of dogs used in research but also to ensure that high-quality dogs are available for research. A high-quality dog for research is a normal, healthy, well-adapted animal.

Abbreviations used in this article: AAHA, American Animal Hospital Association; BCS, body condition score; BP, blood pressure; BW, body weight; CPV, canine parvovirus; ECG, electrocardiogram; GLP, good laboratory practice; HPA, hypothalamic-pituitary-adrenal; HR, heart rate; IACUC, institutional animal care and use committee; ID, interdigital; IV, intravenous; ME, maintenance energy; PVC, polyvinyl chloride; SPF, specific pathogen-free; VOHC, Veterinary Oral Health Council.

References

AAHA [The American Animal Hospital Association]. 2003. Pain Management Standards. From the AAHA Standards of Accreditation: Quality of Care, Pain Management. Lakewood CO: AAHA Press. Available online (www.aahanet.org).

Adams KM, Navarro AM, Hutchinson EK, Weed JL. 2004. A canine socialization and training program at the National Institutes of Health. Lab Anim 33:32-36.

Adams RJ. 2002. Techniques of experimentation In: Fox JG, Anderson LC, Loew FM, Quimby FW, eds. Laboratory Animal Medicine. 2nd ed. New York: Academic Press. p 1013-1015.

Aguila HN, Pakes SP, Lai WC, Lu YS. 1988. The effect of transportation stress on splenic natural killer cell activity in C57BL/6J mice. Lab Anim Sci 38:148-151.

Bayne K. 2002. Development of the human-research animal bond and its impact on animal well-being. ILAR J 43:1-9.

Bayne KAL, Beaver BV, Mench JA, Morton. 2002. Laboratory animal behavior. In: Fox JG, Anderson LC, Loew FM, Quimby FW, eds. Laboratory Animal Medicine. 2nd ed. New York: Academic Press. p 1239-1264.

Beerda B, Schilder MBH, Bernadina W, Van Hooff JARA, DeVries HW, Mol JA. 1999. Chronic stress in dogs subjected to social and spatial restriction. II. Hormonal and immunological responses. Physiol Behav 66:243-254.

Blood DC, Studdert VAP. 1990. Balliere's Comprehensive Veterinary Dictionary. London: Balliere Tindall.

Boxall J, Heath S, Bate S, Brautigam J. 2004. Modern concepts of socialisation for dogs: Implications for their behaviour, welfare and use in scientific procedures. ATLA 32(Suppl 2):81-93.

Burkholder WJ. 2000. Use of body condition scores in clinical assessment of the provision of optimal nutrition. J Am Vet Med Assoc. 217:650-654.

Butterwick RF, Hawthorne AJ. 1998. Advances in dietary management of obesity in dogs and cats. J Nutr 128:2771S-2775S.

BVAAWF/FRAME/RSPCA/UFAW [British Veterinary Association's Animal Welfare Foundation/Fund for Replacement of Animals in Medical Experimentation/Royal Society for Protection of Cruelty to Animals/University Federation for Animal Welfare] Joint Working Group on Refinement. 2001. Morton DB, Jennings M, Buckwell A, Ewband R, Godfrey C, Holgate B, Inglis I, James R, Page C, Sharman I, Verschoyle R, Westall L, Wilson AB. Refining procedures for the administration of substances Report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Lab Anim 35:1-41.

BVAAWF/FRAME/RSPCA/UFAW [British Veterinary Association's Animal Welfare Foundation/Fund for Replacement of Animals in Medical Experimentation/Royal Society for Protection of Cruelty to Animals/University Federation for Animal Welfare] Joint Working Group on Refinement. 2003. Morton DB, Hawkins P, Bevan R, Heath K, Kirkwood J, Pearce P, Scott L, Whelan G, Webb A. Refinements in telemetry procedures. Seventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Part A. Lab Anim 37:261-299.

BVAAWF/FRAME/RSPCA/UFAW [British Veterinary Association's Animal Welfare Foundation/Fund for Replacement of Animals in Medical Experimentation/Royal Society for Protection of Cruelty to Animals/University Federation for Animal Welfare] Joint Working Group on Refinement. 2004a. Hawkins P, Morton, Bevan R, Heath K, Kirkwood, Pearce P, Scott L, Whelan G, Webb A. Husbandry refinements for rats, mice, dogs and non-human primates used in telemetry procedures: Seventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement, Part B Lab Anim 38: 1-10.

BVAAWF/FRAME/RSPCA/UFAW [British Veterinary Association's Animal Welfare Foundation/Fund for Replacement of Animals in Medical Experimentation/Royal Society for Protection of Cruelty to Animals/University Federation for Animal Welfare] Joint Working Group on Refinement. Prescott MJ, Morton DB, Anderson D, Buckwell AC, Heath SE, Hubrecht RC, Jennings M, Robb D, Ruane R, Swallow J, Thompson P. 2004b. Refining dog husbandry and care: Eight report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement, Lab Anim 38(S1):63-66.

Campbell SA, Hughes HC, Griffin HE, Landi MS, Mallon FM. 1988. Some effects of limited exercise on purpose-bred beagles. Am J Vet Res 49:298-301.

CCAC [Canadian Council on Animal Care]. 1993. Guide to the Care and Use of Experimental Animals. Vol 1, Ed 2. Olfert ED, Cross BM, McWilliam AA, eds. Ottawa: CCAC.

CFR [Code of Federal Regulations]. 1978. Good laboratory practice regulation for nonclinical laboratory studies 21 CFR 58. Washington DC: Office of the Federal Register.

CFR [Code of Federal Regulations]. 1985. Title 9 (Animals and Animal Products), Subchapter A (Animal Welfare). Washington DC: Office of the Federal Register.

Chalker VJ, Toomey C, Opperman S, Brooks HW, Ibuoye MA, Brownlie J, Rycroft AN. 2003. Respiratory disease in kennelled dogs: Serological responses to Bordetella bronchiseptica lipopolysaccharide do not correlate with bacterial isolation or clinical respiratory symptoms. Clin Diag Lab Immunol 10:352-356.

Clark JD, Rager DR, Calpin JP. 1997a. Animal Well-Being II. Stress and distress. Lab Anim Sci 47:571-579.

Clark JD, Rager DR, Calpin JP. Animal Well-Being III. 1997b. An overview of assessment. Lab Anim Sci 47:571-579.

Clark JD, Rager DR, Calpin JP. Animal Well-Being IV. 1997c. Specific Assessment Criteria. Lab Anim Sci 47: 586-5797.

Clark RD, Stainer JR. 1994. Medical & Genetic Aspects of Purebred Dogs. Fairway KS: Forum Publications.

Colflesh L. 2004. Making Friends: Training Your Dog Positively. Hoboken NJ: Howell Book House.

DeBowes LJ. 2000. Dentistry: Periodontal aspects. In: Ettinger SJ, Feldman EC, eds. Textbook of Veterinary Internal Medicine Diseases of the Dog and Cat. Vol 2, Ed 5. Philadelphia: WB Saunders. p 1127-1134.

DeBowes LJ, Mosier D, Logan E, Harvey CE, Lowry S, Richardson DC. 1996. Association of periodontal disease and histologic lesions in multiple organs from 45 dogs. J Vet Dent 13:57-60.

Disney's Animal Kingdom Animal Training website (www.animaltraining.org).

Donaldson J. 1996. The Culture Clash. Berkley: James and Kenneth.

Dorsten, DM, Cooper, DM. 2004. Use of body condition scoring to manage body weight in dogs. Contemp Top 43:34-37.

Drozdowicz CK, Bowman TA, Webb ML, Lang CM. 1990. Effect of in-house transport on murine plasma corticosterone concentration and blood lymphocyte populations. Am J Vet Res 51:1841-1846.

Dugan JP, Meunier LD. 1996. A Positive Reinforcement Training program for Restraint of Dogs. Presented at the Delaware Valley Branch (DVB) AALAS meeting held June 1996 in Philadelphia, Pennsylvania.

DuPont GA 1998. Prevention of periodontal disease. Vet Clin N Am Small Anim Pract Vet 28:1129-1145.

Duquette RA, Nuttall TJ. 2004. Methicillin-resistant Staphylococcus aureus in dogs and cats: An emerging problem? 45:587-588.

Dysko RC, Nemzek JA, Levin SI, DeMarco GJ, Moalli MR. 2002. Biology and diseases of dogs. In: Fox JG, Anderson LC, Loew FM, Quimby FW, eds. Laboratory Animal Medicine. 2nd ed. New York: Academic Press. p 395-458.

Fillman-Holliday D, Landi MS. 2002. Animal care best practices for regulatory testing. ILAR J 43:S49-S58.

Fox MW. 1966. The development of learning and conditioned responses in the dog: Theoretical and practical implications. Can J Comp Med Vet Sci 30:282-286.

Fox MW. 1978. The Dog: Its Domestication and Behaviour. New York: Garland Stamp Press.

Fox MW, Stelzner D. 1966. Approach/withdrawal variables in the development of social behaviour in the dog. Anim Behav 14:362-366.

Gaebelein CJ, Calosy R, Boticelli L, Howard JL, Obrist PA. 1977. Blood pressure and cardiac change during signalled and unsignalled avoidance in dogs. Physiol Behav 19:69-74.

Galosy RA, Clarke LK, Mitchell JH. 1979. Cardiac changes during behavioural stress in dogs. Am J Physiol. 236:H750-H758.

Gantt WH, Newton JEO, Royer FL, Stephen JH. 1966. Effect of person. Cond Reflex 1:18-35.

Gorrel C, Bierer TL. 1999. Long term effects of a dental hygiene chew on the periodontal health of dogs. J Vet Dent 16:109-113.

Grandin T. 1997. Assessment of stress during handling and transport. J Anim Sci 75:249-257.

Greene CE. 2000. Bacterial diseases. In: Ettinger SJ, Feldman EC, eds. Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. Vol 1, Ed 5. Philadelphia: WB Saunders. p 391-392.

Greene CE, Schultz RD, Ford RB. 2001. Canine vaccination. Vet Clin N Am Small Anim Pract Vet 31:473-492.

Harvey CE. 1998. Periodontal disease in dogs: Etiopathogenesis, prevalence, and significance. Vet Clin N Am Small Anim Pract 28:1111.

Harvey CE, Emily PP. 1993. Small Animal Dentistry. St. Louis: Mosby. p 102-103.

Hawkins P, Morton DB, Bevan R, Heath K, Kirkwood J, Pearce P, Scott L, Whelan D, Webb A. (Members of the Joint Working Group on Refinement). 2004. Husbandry refinements for rats, mice, dogs and non-human primates used in telemetry procedures. Seventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement, Part B. Lab Anim 38:1-10

Hefferren JJ, Schiff TG, Smith MR. 1994. Assessment methods and clinical outcomes: Chemical and microbial composition, formation, and maturation dynamics of pellicle, plaque, and calculus. J Vet Dent 11:75-79.

Hendrick MJ, Dunagan CA. 1991. Focal necrotizing Granulomatous panniculitis associated with subcutaneous injection of rabies vaccine in cats and dogs: 10 cases (1988-1989). J Am Vet Med Assoc 198:304-305.

Hennessy MB, Williams MT, Miller DD, Douglas CW, Voith VL. 1998. Influence of male and female petters on plasma cortisol and behaviour: Can human interaction reduce the stress of dogs in a public animal shelter? Appl Anim Behav Sci 61:63-77.

Hetts S. 1991. Psychologic well-being: Conceptual issues, behavioral measures, and implication for dogs. Vet Clin N Am Small Anim Pract 21:369-387.

Hetts S, Clark JD, Calpin JP, Arnold CE, Mateo JM. 1992. Influence of housing condition on beagle behaviour. Appl Anim Behav Sci 34:137-155.

Heusner AA. 1991. Body mass, maintenance and basal metabolism in dogs. J Nutr 121:S8-S17.

Hogenesch H, Azcona-Olivera J, Scott-Moncrieff C, Snyder PW, Glickman LT. 1999. Vaccine-induced autoimmunity in the dog. Adv Vet Med 41:715-732.

Holmstrom SE, Fitch PF, Eisner ER. 2004. Veterinary Dental Techniques for the Small Animal Practitioner. 3rd ed. Philadelphia: WB Saunders.

Horwitz D, Mills D, Heath S. 2002 BSAVA Manual of Canine and Feline Behavioural Medicine. Gloucester: British Small Animal Veterinary Association.

Hoskins JD. 2000. Canine viral diseases. In: Ettinger SJ, Feldman EC eds. Textbook of Veterinary Internal Medicine Disease of the Dog and Cat. 5th ed. Philadelphia: WB Saunders.

Hubrecht RC. 1995. Enrichment in puppyhood and its effects on later behavior of dogs. Lab Anim Sci 45:70-75.

Hubrecht RC, Serpell, Poole TB. 1992. Correlates of pen size and housing conditions on the behaviour of kennelled dogs. Appl Anim Behav Sci 34:365-383.

Hughes HC, Campbell S. 1990. Effects of primary enclosure size and human contact. In: Mench JA, Krulisch L, eds. Canine Research Environment. Bethesda: Scientists Center for Animal Welfare. p 66-73.

IATA [International Air Transport Association]. 2000. Live Animals Regulations. 27th ed. 1 October 2000. Montreal: IATA.

Joles JA, den Hertog JM, Huisman GH, Kraan WJ, van Schaik FW, Schrikker ACM. 1982. Plasma renin activity and plasma catecholamines in intact and splenectomized running and swimming beagle dogs. Eur J Appl Physiol 49:111-119.

Kemming GI, Messick JB, Enders G, Boros M, Lorenz B, Muenzing S, Kisch-Wedel H, Mueller W, Hahmann-Mueller A, Messmer K, Thein E. 2004a. Mycoplasma haemoncanis infection—A kennel disease? Comp Med 54:404-409.

Kemming G, Messick JB, Mueller W, Enders G, Meisner F, Muenzing S, Kisch-Wedel H, Schropp A, Wojtczyk C, Packert K, Messmer K, Thein E. 2004b. Can we continue research in splenectomized dogs? Mycoplasma haemoncanis: Old problem-new insight. Eur Surg Res 36:198-205.

Kissinger JT, Garver EM, Schnell MA, Schantz JD, Coatney RW, Meunier LD. 1998. A new method to collect bile and access the duodenum in conscious dogs. Contemp Top 37:89-93.

Klingborg DJ, Hustead DR, Curry-Galvin EA, Gumley NAAR, Henry SC, Fairfield TB, Paul MA, Boothe DM, Shawn Blood K, Huxsoll DL, Reynolds DL, Gatz Riddell M, Reid JS, Short CR. 2002. AVMA Council on Biologic and Therapeutic Agents' report on cat and dog vaccines. J AVMA 221:1401-1407.

Kovacs MS, McKiernan S, Potter DM, Chilappagari S. 2005. An epidemiological study of interdigital cyst in a research beagle colony. Contemp Top 44:17-21.

Kuhn G, Lichtwald K, Hardegg W., Abel HH. 1991. The effect of transportation stress on circulating corticsteroids, enzyme activities and hematological values in laboratory dogs. J Exp Anim Sci 34:99-104.

Kwei GY, Gehret JR, Novak LB, Drag MD, Goodwin T. 1995. Chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs. Lab Anim Sci 45:683-685.

Laflamme DP. 1997. Development and validation of a body condition score system for dogs. Canine Pract 22:10-15.

Landi MS, Kissinger JT, Campbell SA, Kenney CA, Jenkins EL. 1990. The effects of four types of restraint on serum alanine aminotransferase and aspartate aminotransferase in the Macaca fascicularis. J Am Coll Toxicol 9:517-523.

Landi MS, Kreider JW, Lang CM. 1982. Effects of shipping on the immune function in mice. Am J Vet Res 43:1654-1657.

Laule GE. 2003. Positive reinforcement training and environmental enrichment: Enhancing animal well-being. JAVMA 223:969-973.

Lindsay SR. 2000. Handbook of Applied Dog Behavior and Training. Vol I. Adaption and Learning. Ames: Iowa State Press.

Lindsay SR. 2001. Handbook of Applied Dog Behavior and Training. Vol II. Etiology and Assessment of Behavior Problems. Ames: Iowa State Press.

Lindsay SR. 2005. Handbook of Applied Dog Behavior and Training. Vol III. Procedures and Protocols. Ames: Blackwell Publishing.

Loe H. 1967. The gingival index, the plaque index and the retention index systems. J Periodon 38:610-616.

Logan EI, Boyce EN. 1994. Oral health assessment in dogs: Parameters and methods. J Vet Dent 11:58-63.

Marder A, Reid PJ. 1996. Treating canine behaviour problems: Behaviour modification, obedience and agility training. In: Voith JL, Borchelt PL, eds. Readings in Companion Animal Behavior. p 56-61.

McFarland D. 1981. The Oxford Companion to Animal Behavior. New York: Oxford University Press.

Mann WA, Landi MS, Horner E, Woodward P, Campbell S, Kinter LB. 1987. A simple procedure for direct blood pressure measurements in conscious dogs. Lab Anim Sci 37:105-108.

Medleau L, Hnilica KA. 2006. Small Animal Dermatology: A Color Atlas and Therapeutic Guide. 2nd ed. St. Louis: WB Saunders.

Meunier LD, d'Epagnier DL, Morasco KO, Caplan H, Holliday DF. 2004. Identification of Plesiomonas shigelloides and Campylobacter spp. in Colony-Bred Beagles. National AALAS Meeting held in Seattle, Washington, October 2004.

Meunier LD, Kissinger JT, Marcello J, Nichols AJ, Smith PL. 1993. A chronic access port model for direct delivery of drugs into the intestine of conscious dogs. Lab Anim Sci 43:466-470.

Morris JG, Rogers QR. 2000. Nutrition of healthy dogs and cats in various stages of adult life. In: Ettinger SJ, Feldman EC, eds. Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. Vol 1, Ed 5. Philadelphia: WB Saunders. p 236-240.

Morris JG, Rogers QR, Fascetti AJ. 2005. Nutrition of healthy dogs and cats in various stages of adult life. In: Ettinger SJ, Feldman EC, eds. Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. Vol 1, Ed 5. Philadelphia: WB Saunders. p 555-560.

Mouzin DE, Lorenzen MJ, Haworth JD, King VL. 2004. Duration of serologic response to five viral antigens in dogs. J AVMA 224:55-60.

NIH [National Institutes of Health]. 2002. Public Health Service Policy on Humane Care and Use of Laboratory Animals. Washington DC: US Department of Health and Human Services.

NRC [National Research Council]. 1991. Education and Training in the Care and Use of Laboratory Animals. A Guide for Developing Institutional Programs. Washington DC: National Academy Press.

NRC [National Research Council]. 1992. Recognition and Alleviation of Pain and Distress in Laboratory Animals. Washington DC: National Academy Press.

NRC [National Research Council]. 1994. Laboratory Animal Management Dogs. Washington DC: National Academy Press. NRC [National Research Council]. 1996. Guide for the Care and Use of Laboratory Animals. 7th ed. Washington DC: National Academy Press.

NRC [National Research Council]. 1996. Guide for the Care and Use of Laboratory Animals. 7th ed. Washington DC: National Academy Press.

NRC [National Research Council]. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington DC: National Academies Press.

Odendall JSJ, Meintjes RA. 2003. Neurophysiological correlates of affiliative behaviour between humans and dogs. Vet J 165:296-301.

Overall KL. 1997. Clinical Behavioral Medicine for Small Animals. St. Louis: Mosby.

Overall KL. 2000. Natural animal models of human psychiatric conditions: Assessment of mechanism and validity. Prog Neuro-Psychopharm Biol Psychiatry 24:727-776.

Overall KL, Dyer K. 2005. Enrichment strategies for laboratory animals from the viewpoint of clinical veterinary behavioral medicine: Emphasis on cats and dogs. ILAR J 46:202-216.

Paul MA, Appel M, Barrett R, Carmichael LE, Childers H, Cotter S. Davidson A, Ford R, Keil D, Lappin M, Schultz RD, Thacker E, Trumpeter, Welborn L. 2003. Report of the American Animal Hospital Association (AAHA) Canine Vaccine Task Force: 2003 Canine Vaccine Guidelines, Recommendations, and Supporting Literature.

Paul MA, Carmichael LE, Childers H, Cotter S, Davidson A, Ford R, Hurley KF, Roth JA, Schultz RD, Thacker E, Welborn L. 2006. Guidelines. J Am Anim Hosp Assoc 42:80-89.

Pongracz P, Miklosi A, Kubinyi E, Topal J, Csanyi V. 2003. Interaction between individual experience and social learning in dogs. Anim Behav 65:595-603.

Pryor, K. 1999. Don't Shoot the Dog. The New Art of Teaching and Training. New York: Bantam Books.

Raekallio MR, Kuusela EK, Lehtinen ME, Tykkylainen MK, Huttunen P, Westerholm FC. 2005. Effects of exercise-induced stress and dexamethasone on plasma hormone and glucose concentrations and sedation in dogs treated with dexmedetomidine. Am J Vet Res 66:260-265.

Reeve EB, Gregerson MI, Allen TH, Sear H. 1953. Distribution of cells and plasma in the normal and splenectomised dog and its influence on blood volume with P and T-1824. Am J Physiol 175:195-203.

Reilly J. 1998. Variables in animal based research: Part 2. Variability associated with experimental conditions and techniques. ANZCCART News 11, Insert 1-12. Available online (http://www.adelaide.edu.au/ANZCCART/).

Remillard RL. 2005. Obesity: A disease to be recognized and managed. In: Ettinger SJ, Feldman EC, eds. Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. Vol 1, Ed 6. Philadelphia: Elsevier Saunders. p 76-78.

Richardson DC, Zicker SC. 2000. Developmental orthopedic disease of dogs. In: Ettinger SJ, Feldman EC, eds. Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. Vol 1, Ed 5. Philadelphia: WB Saunders. p 245-251.

Sato N, Shen YT, Kiuchi K, Shannon RP, Vatner SF. 1995. Splenic contraction-induced increases in arterial O₂ reduce requirement for CBF in conscious dogs. Am J Physiol 68:H491-H502.

Scott JP, Fuller JL. 1965. Genetics and the Social Behavior of the Dog. Chicago: University of Chicago Press.

Schultz RD. 2000. Considerations in Designing Effective and Safe Vaccination Programs for Dogs. In: Carmichael LE, ed. Recent Advances in Canine Infectious Diseases. International Veterinary Information Service (www.ivis.org)

Shoenfeld Y, Aron-Maor. 2000. Vaccination and autoimmunity—“Vaccinosis”: A dangerous liaison? J Autoimmun 14:1-10.

Shull-Selcer E, Stagg W. 1991. Advances in the understanding and treatment of noise phobias. Vet Clin N Am Small Anim Pract 21:353:367.

Slaughter MR, Birmingham JM, Patel B, Whelan GA, Krebs-Brown AJ, Hockings PD, Osborne JA. 2002. Extended acclimatization is required to eliminate stress effects of periodic blood-sampling procedures on vasoactive hormones and blood volume in beagle dogs. Lab Anim 36:403-410.

Stasiak KL, Maul D, French E, Hellyer PW, Vandewoude S. 2003. Species-specific assessment of pain in laboratory animals. Contemp Top 42:13-20.

Swallow J, Anderson D, Buckwell AC, Harris T, Hawkins P, Kirkwood J, Lomas M, Meacham S, Peter A, Prescott M, Owen S, Quest R, Sutcliffe R, Thompson K. 2005. Guidance on the transport of laboratory animals: Report of the Transport Working Group established by the Laboratory Animal Science Association (LASA). Lab Anim 39:1-39.

Tilley LP, Smith FWK, eds. 2000. Campylobacterosis. In: The 5-Minute Veterinary Consult Canine and Feline. 2nd ed. Baltimore: Lippincott Williams & Wilkins. p 514-515.

Tuli JS, Smith JA, Morton DB. 1995. Stress measurement in mice after transportation. Lab Anim 29:132-138.

Turner PA, Smiler KL, Hargaden M, Koch MA. 2003. Refinements in the care and use of animals in toxicology studies—Regulation, validation, and progress. Contemp Top 42:8-15.

Van den Berg L, Schilder MBH, Knol BW. 2003. Behavior genetics of canine aggression: Behavioral phenotyping of golden retrievers by means of an aggression test. Behav Genet 33:469-483.

Wiggs RB, Lobprise WB 1997. Veterinary Dentistry: Principles and Practice. Philadelphia: Lippincott-Raven.

Wolfle TL. 1987. Control of stress using non-drug approaches. J Am Vet Med Assoc 191:1219-1221.

Wolfle TL. 1990. Policy, program and people: The three P's to well-being. In: Mench JA, Krulisch L, eds. Canine Research Environment. Bethesda: Scientists Center for Animal Welfare. p 41-47.